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The CD8 memory T cell repertoire to the influenza A derived M1(58-66) epitope shows a restricted V genes and CDR3 sequences usage. The repertoire is highly polyclonal and the clonotype distribution has been described as consisting of two components, one showing a power law-like distribution and the other composed of a few clonotypes with a very high relative frequency. The question is whether the complex repertoire defined by its ability to flourish in a short term recall culture corresponded to functional cells. Here we show that there is a relation between expression of the degranulation marker CD107 and cytotoxicity or IFN-γ production in CD8 T cell lines and clones. We then examine recently degranulated CD8 cells from recall cultures from four middle aged HLA-A2 subjects and show that these functional cells are polyclonal. The clonotype distributions of the CD8(+)CD107(+) repertoires are complex in the same manner as previously reported. The clonotype composition of CD8(+)CD107(+) repertoires is also very similar to CD8 only repertoires, and to CD8(+)HLA-A2-M1(58-66) pentamer positive repertoires. We postulate that multiple exposures during childhood to this conserved influenza A epitope has generated a complex functional repertoire in HLA-A2 individuals.

We report phenotypic and genetic analyses of a recessive, larval lethal zebrafish mutant, bal(a69), characterized by severe eye defects and shortened body axis. The bal(a69) mutation was mapped to chromosome 24 near the laminin alpha 1 (lama1) gene. We analyzed the lama1 gene sequence within bal(a69) embryos and two allelic mutants, bal(arl) and bal(uw1). Missense (bal(a69)), nonsense (bal(arl)), and frameshift (bal(uw1)) alterations in lama1 were found to underlie the phenotypes. Extended analysis of bal(a69) ocular features revealed disrupted lens development with subsequent lens degeneration, focal cornea dysplasia, and hyaloid vasculature defects. Within the neural retina, the ganglion cells showed axonal projection defects and ectopic photoreceptor cells were noted at inner retinal locations. To address whether ocular anomalies were secondary to defects in lens differentiation, bal(a69) mutants were compared to embryos in which the lens vesicle was surgically removed. Our analysis suggests that many of the anterior and posterior ocular defects in bal(a69) are independent of the lens degeneration. Analysis of components of focal adhesion signaling complexes suggests that reduced focal adhesion kinase activation underlies the anterior segment dysgenesis in lama1 mutants. To assess adult ocular phenotypes associated with lama1 mutations, genetic mosaics were generated by transplanting labeled bal cells into ocular-fated regions of wild-type blastulas. Adult chimeric eyes displayed a range of defects including anterior segment dysgenesis and cataracts. Our analysis provides mechanistic insights into the developmental defects and ocular pathogenesis caused by mutations in laminin subunits.

The human PITX3 gene encodes a bicoid-like homeodomain transcription factor associated with a variety of congenital ocular conditions, including anterior segment dysgenesis, Peter's anomaly, and cataracts. We identified a zebrafish pitx3 gene encoding a protein (Pitx3) that possesses 63% amino acid identity with human PITX3. The zebrafish pitx3 gene encompasses approximately 16.5 kb on chromosome 13 and consists of four exons, which is similar to the genomic organization of other pitx genes. Expression of the zebrafish pitx3 gene was studied by in situ mRNA hybridization and RT-PCR. The pitx3 transcripts were detected throughout development with the greatest level of expression occurring in the developing lens and brain at 24 hpf. In adults, the highest expression was detected in the eye. Morpholinos were used to knockdown expression of the Pitx3 protein and a control morpholino that contains five mismatched bases was used to confirm the specificity of the phenotypes. The morphants had small eyes, misshapen heads and reduced jaws and fins relative to controls. The morphants exhibited abnormalities in lens development and their retinas contained pyknotic nuclei accompanied by a reduction in the number of cells in different neuronal classes. This suggests the lens is required for retinal development or Pitx3 has an unexpected role in retinal cell differentiation or survival. These results demonstrate zebrafish pitx3 represents a true ortholog of the human PITX3 gene and the general function of the Pitx3 protein in lens development is conserved between mammals and the teleost fish.

Coordination of rhythmic locomotion depends upon a precisely balanced interplay between central and peripheral control mechanisms [1]. Although poorly understood, peripheral proprioceptive mechanosensory input is thought to provide information about body position for moment-to-moment modifications of central mechanisms mediating rhythmic motor output [2]. Pickpocket1 (PPK1) is a Drosophila subunit of the epithelial sodium channel (ENaC) family displaying limited expression in multiple dendritic (md) sensory neurons tiling the larval body wall and a small number of bipolar neurons in the upper brain [3]. ppk1 null mutant larvae had normal external touch sensation and md neuron morphology but displayed striking alterations in crawling behavior. Loss of PPK1 function caused an increase in crawling speed and an unusual straight path with decreased stops and turns relative to wild-type. This enhanced locomotion resulted from sustained peristaltic contraction wave cycling at higher frequency with a significant decrease in pause period between contraction cycles. The mutant phenotype was rescued by a wild-type PPK1 transgene and duplicated by expressing a ppk1RNAi transgene or a dominant-negative PPK1 isoform. These results demonstrate that the PPK1 channel plays an essential role in controlling rhythmic locomotion and provide a powerful genetic model system for further analysis of central and peripheral control mechanisms and their role in movement disorders.

How the naive T cell repertoire arises and forms the memory repertoire is still poorly understood. This relationship was analyzed by taking advantage of the focused TCR usage in HLA-A2–restricted CD8 memory T cell responses to influenza M158–66. We analyzed rearranged BV19 genes from CD8 single-positive thymocytes, a surrogate for the naive repertoire, from 10 HLA-A2 individuals. CDR3 amino acid sequences associated with response to influenza were observed at higher frequencies than expected by chance, an indicator of preselection. We propose that a rearrangement mechanism involving long P-nucleotide addition from the J2.7 region explains part of this increase. Special rearrangement mechanisms can result in identical T cells in different individuals, referred to as public responses. Indeed, the rearrangements utilizing long P nucleotide additions were commonly observed in the response to the M158–66 epitope in 30 HLA-A2 middle-aged adults. Thus, in addition to negative and positive selection, special rearrangement mechanisms may influence the composition of the naive repertoire, resulting in more robust responses to a pathogen in some individuals.

BackgroundLaminins represent major components of basement membranes and play various roles in embryonic and adult tissues. The functional laminin molecule consists of three chains, alpha, beta and gamma, encoded by separate genes. There are twelve different laminin genes identified in mammals to date that are highly homologous in their sequence but different in their tissue distribution. Thelaminin alpha -1gene was shown to have the most restricted expression pattern with strong expression in ocular structures, particularly in the developing and mature lens.ResultsWe identified the zebrafishlama1gene encoding a 3075-amino acid protein (lama1) that possesses strong identity with the human LAMA1. Zebrafishlama1transcripts were detected at all stages of embryo development with the highest levels of expression in the developing lens, somites, nervous and urogenital systems. Translation of thelama1gene was inhibited using two non-overlapping morpholino oligomers that were complementary to sequences surrounding translation initiation. Morphant embryos exhibited an arrest in lens development and abnormalities in the body axis length and curvature.ConclusionThese results underline the importance of thelaminin alpha 1for normal ocular development and provide a basis for further analysis of its developmental roles.

The effect of nutritional stress on phenotypical and genetic variation was examined for five morphological traits (thorax length, wing length, sternopleural chaeta number, abdominal chaeta number and arista branch number) in 30 isofemale lines of Drosophila melanogaster. Phenotypical variation of all traits except sternopleural chaeta number and fluctuating asymmetry of all bilateral traits were significantly higher in flies reared under poor feeding conditions. Estimates of isofemale line heritability (coefficients of intraclass correlation) did not show a consistent pattern among traits. However, additive genetic variance was generally higher in poor feeding conditions in all traits except sternopleural chaeta number, although these differences were not statistically significant. Similarly, estimates of evolvability were higher under nutritional stress for all traits except sternopleural chaeta number. These results suggest that nutritional stress increases the expression of genetic variation for some morphological traits in Drosophila and, in this respect, is similar to the effects of temperature stress studied previously.

Axenfeld-Rieger syndrome (ARS) is a relatively rare developmental disorder characterized by maxillary hypoplasia, anterior segment defects of the eye (with glaucoma in ∼50%), and dental and umbilical abnormalities. The PITX2 homeodomain-containing transcription factor gene plays a major role in this condition, explaining approximately 40% of classic ARS.1–5 In addition, PITX2 mutations were shown to cause isolated ocular conditions,6–8 to be associated with additional brain anomalies,9,10 and possibly to contribute to other phenotypes such as omphalocele and VATER-like association.11 The function of Pitx2 is conserved in vertebrates. Total or conditional knockouts of Pitx2 result in severe developmental phenotypes in mice that include craniofacial, ocular, dental, brain, heart, lung, and other systemic defects consistent with the expression sites of Pitx2. With respect to Axenfeld-Rieger syndrome, Pitx2-deficient mice exhibit arrest of eye and tooth organogenesis, defective body wall closure and brain abnormalities.12–17 The defects observed in mice are associated with a complete loss of Pitx2 activity in the corresponding structures and, therefore, are noticeably more severe that what is reported in humans with heterozygous PITX2 mutations, whereas heterozygous mice are generally described as normal. This suggests a variable requirement of PITX2/Pitx2 for normal human/mouse embryogenesis, with human development more sensitive to correct PITX2 dosage. Human PITX2 mutations mainly result in a complete or partial loss of function, with mutations retaining some wild-type activity producing milder phenotypes.18–20 Some mutations identified in patients with Axenfeld-Rieger syndrome displayed very minor defects in DNA-binding and transactivation activities, again suggesting that normal human development is highly susceptible to alterations of PITX2 function.5 In addition, several human mutations associated with gain-of-function have been reported,21 consistent with the dramatic ocular and limb phenotypes seen in mice with Pitx2 overexpression during eye and forelimb development.22,23 Therefore, correct dosage of PITX2 is critical for normal development, which further underscores the importance of precise transcriptional regulation of its expression. Regulation of Pitx2 expression has been studied by several groups, but only a few cis-regulatory elements have been identified and examined in animal models. In mouse, Pitx2 appears to be a part of the Wnt/β-catenin/TCF/LEF and cAMP-CREB signaling pathways.24,25 Transcriptional activation of Pitx2 during eye development has been shown to be dependent on retinoic acid signaling26–28; the nature of this interaction, direct or indirect, has not yet been established. A few studies reported in vivo identification of Pitx2 enhancer elements, which facilitated upstream factor analysis. Asymmetric Pitx2 expression in the developing visceral organs was shown to require an intronic enhancer located in the proximity of the last exon,29,30 whereas a 7-kb fragment located 4.2 kb downstream of the last exon of Pitx2 was found to direct reporter expression in the oral ectoderm and Rathke's pouch.31 Finally, several regulatory elements associated with pitx2 expression in the stomodeum territory have been isolated in ascidians; the cis-sequences were either intronic or located within 2.5 kb of the 5′ or 3′ end of the gene.32 PITX2 is flanked by glutamyl aminopeptidase (aminopeptidase A) (ENPEP) located approximately 54 kb downstream and chromosome 4 open reading frame 32 (C4orf32) expressed sequence positioned approximately 1.5 Mb upstream of PITX2; thus, the 1.5-Mb sequence upstream of the gene is considered a gene desert. Gene deserts often contain conserved sequences involved in transcriptional regulation of nearby genes.33 The presence of PITX2 regulatory elements in this region were consistent with the previous reports of patients with Axenfeld-Rieger syndrome with translocation breakpoints that occurred within the distant upstream region and did not disrupt the coding region of PITX2.1,2,34,35 We describe identification of several novel regulatory sequences in the PITX2/pitx2 region, including several distant elements located within the gene desert upstream of PITX2. The activities of these elements appear to be consistent with PITX2/pitx2 expression, suggesting that they play a role in this gene's function. In addition, we report the identification of a de novo deletion located 106 to 108 kb upstream of PITX2 in a patient with Axenfeld-Rieger syndrome, thus providing additional support for the essential role of the identified regulatory elements in PITX2 function.